Recent advances in the understanding of T-cell activation have led to new therapeutic approaches in the treatment of immunological disorders. One attractive target of intervention has been the blockade of T cell-mediated co-stimulatory pathways, which result in more selective effects on only those T-cells that have encountered specific antigen. In fact, in some instances, CD28/B7 co-stimulatory pathway antagonists can induce antigen-specific tolerance that prevents the progression of autoimmune diseases and organ graft rejection. However, the negative regulatory role of CTLA-4 in autoimmune diseases and graft rejection supported a dynamic but complex process of immune regulation prominent in the control of self-reactivity. As an example, CD28 and CTLA-4 can regulate pathogenic as well as suppressor cell development and function resulting, paradoxically, in opposite function consequences of receptor-ligand interactions. Finally, unlike CD28, the biochemical signals initiated through the CTLA-4 pathway are biochemically-linked to the T cell receptor signaling-complex. Thus, we hypothesize that CTLA-4 plays a critical role in intrinsic regulation of the ongoing autoimmune response by influencing the activation threshold of the T cells resulting in inactivation or altered differentiation. Moreover, manipulation of immune responses through CTLA-4 will depend on effective co-ligation of the TCR and CTLA-4. In order to test this hypothesis, the following specific aims are proposed. 1. TO DETERMINE THE MOLECULAR AND BIOCHEMICAL BASIS OF CTLA-4-MEDIATED T CELL FUNCTION IN IMMUNE TOLERANCE; 2. TO DETERMINE THE ROLE OF CTLA-4/B7 INTERACTIONS IN REGULATION OF PATHOGENESIS AND TOLERANCE INDUCTION AND MAINTENANCE IN NOD DIABETES; 3. TO DETERMINE THE ROLE OF CTLA-4 ON REGULATORY T CELLS THAT SUPPRESS NOD DIABETES; AND 4. TO USE A SINGLE CHAIN ANTI-CTLA-4 AND pepMHC-IG DIMER TO PRODUCE A NOVEL DIMERIC pepMHC-IG/ANTI-CTLA-4 HETEROCONJUGATE TO SPECIFICALLY TOLERIZE AUTOREACTIVE T CELLS. The studies proposed will be complimented by the other projects within this program project grant. We will work closely with Dr. Abbas to understand the role of CTLA-4 engagement at different stages of the immune response and solicit the collaborative efforts of Drs. Weiss and DeFranco to elucidate the detailed biochemical events that result from CTLA-4 engagement at the TCR complex. The results of these studies will provide important insights into the mechanism of CD28/CTLA-4 regulation of immune responses and may lead to novel immunotherapeutic approaches to the induction and maintenance of peripheral tolerance.